Large scale cell culture system for making meat and associated products

ABSTRACT

Provided is a large-scale cell culture system for producing products without harming animals. Also provided is a method for making meat products using this cell culture system. Further provided is a method for making personal care products using this cell culture system, as well as a method for making nutritional supplements using this cell culture system.

FIELD OF INVENTION

Described herein is a large scale cell culture system, as well asmethods of making and using it to form engineered meat products,nutritional supplements and personal care compositions. An importantelement of the invention is that the culture system does not require theuse of serum (e.g. fetal bovine serum) an animal derived substance.

BACKGROUND

The human body needs nutrients to be delivered to each of its cells.Once food is decomposed into digestible and non-digestible components,the digestible components are subsequently processed into micronutrientsand eventually delivered to the various organs, tissues and cells mainlyvia the circulatory system. The complex molecules, such as growthfactors, cytokines, etc. that are also needed for the healthyfunctioning of the organism are produced by the organism itself from thebasic nutrients processed from the food intake. This way the organismrepresents a highly autonomous system.

This system is mimicked in the organ-on-the-chip device, amicrophysiological system, where organoids prepared from specializedcells and modeling the various organs of the body are coupled throughmicrofluidic channels representing the circulatory system that carriesthe nutrient-containing culture medium supplied from the exterior. Theseorgan-on-the-chip devices are used as toxicology assays in drugdevelopment to supplement animal models and hoped one day to replaceanimal trials. The organoids used in these devices (prepared separatelyand then inserted into the device) are miniature representations oftissues and organs with linear dimensions typically on the order of afew hundred microns.

The cell culture medium circulated (i.e. pumped) in themicrophysiological circuit contains a multitude of molecules necessaryfor the healthy maintenance and growths of cells. An important componentof such medium is typically serum such as fetal bovine serum (FBS), calfserum, or horse serum. The sera, in particular PBS, require theslaughtering of animals. FBS is also expensive and shows wide variationfront animal to animal.

The nutritional benefits of meat are tempered by potential associatedenvironmental degradation. According to a 2006 report by the Livestock,Environment And Development Initiative, entitled Livestock's LongShadow—Environmental Issues and Options, the livestock industry is oneof the largest contributors to environmental degradation worldwide.Modern practices of raising animals for food contributes widely to airand water pollution, land degradation, climate change, and loss ofbiodiversity. The production and consumption of meat and other animalsources of protein is also associated with the clearing of rainforestsand species extinction. This has led to a significant effort to developclean meat. As used herein, clean meat means providing meat withoutharming animals. Most efforts are based on growing and culturingmammalian cells that require growth medium with components derived fromslaughtered animals for example, fetal bovine serum. Accordingly, thereis a need for an alternative growth method for meat produced withoutharming animals.

Proteins are also used in personal care applications and nutritionalsupplements. For example, proteins are applied to the face to improvethe appearance of skin. Protein bars and protein powders are ingested asnutritional supplements. There is a continuing need for proteinsolutions for personal care and alternatives for nutritionalsupplements.

The inventors have previously described engineered meats and methods ofmaking engineered meats using cultured cells. See, e.g., U.S. Pat. No.8,703,216, titled “ENGINEERED COMESTIBLE MEAT,” hereby incorporated byreference in its entirety. However, bio-manufacturing processes aimed atbuilding extended tissue constructs that require large numbers ofadherent cells face the difficulty of growing these cells (to thebillions to trillions) efficiently and cost effectively. Additionally,the medium required to grow the cells typically include fetal bovineserum which is frowned upon for the reasons cited above and may also beexpensive.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide a large-scale cellculture system for producing products without harming animals. Anotherobjective of the present invention is to provide a method for makingmeat products using this cell culture system. Yet another objective ofthe present invention is to provide a method for making personal careproducts using this cell culture system. Finally, an objective of thepresent invention is to provide a method for making nutritionalsupplements using this cell culture system.

The above objectives highlight certain aspects of the invention.Additional aspects and embodiments of the invention are found in thefollowing detailed description of the invention.

FIGURES

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same become betterunderstood by reference to the following Figures in conjunction with thedetailed description below.

FIG. 1 shows an example of a large-scale cell culture system accordingto the present invention, wherein different cell types are cultured inseparate culturing, vessels and the culturing vessels are connected inseries.

FIG. 2 shows an example of a large-scale cell culture system accordingto the present invention, wherein different cell types are cultured inseparate culturing vessels and the culturing vessels are connected inparallel.

FIG. 3 shows an example of a large-scale combination cell culture systemaccording to the present invention, wherein different cell types arecultured in separate culturing vessels where some are connected inseries (e.g., muscle cells and adipose cells) and others are connectedin parallel (e.g., liver cells and cardiac cells) within the samesystem.

FIG. 4 shows an example of a large-scale cell culture system accordingto the present invention, wherein different cell types are cultured in asingle culturing vessel.

DETAILED DESCRIPTION

Unless specifically defined, all technical and scientific terms usedherein have the same meaning as commonly understood by a skilled artisanin enzymology, biochemistry, cellular biology, molecular biology, andthe medical sciences.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The cell culture system of the present invention includes one or morevessels.

In an embodiment of the invention, a single culturing vessel may be usedto culture the various cell types together. As shown in FIG. 1,culturing vessel 1 contains adipose, muscle and liver cells. Vessel 1 isconnected to fresh medium vessel 2 and waste vessel 3 by line 4. Mediumis pumped through the lines into the vessels with pump 5. Valves 6controls the flow to the vessels, when the valve to the fresh mediumvessel is open, the valve to the waste vessel is closed and visa versa.This approach may make the cells function better than when culturedindependently, far example, by secreting their relevant growth factorsmore efficiently.

It is envisioned within the present invention that when two or moreculturing vessels are used they are connected in parallel or in seriesor a combination of the two.

The vessels may also be referred to as bioreactors. The system allowsculturing of one or several cell types (each in its own culturingvessel) at the same time. Alternatively, several cell types may becultured in a single culturing vessel either as part of a mixture orindependently cultured in isolation separated by partitions orseparators. The cell types may be of different genus and/or species ormay be of the same genus and species, but belong to different sub-types.Alternatively, the cell types may be of the same genus and species, butdiffer from each other based on the biopsy they are obtained from.

As used herein, “vessels connected in parallel” means two or moreculturing vessels are fed from a manifold at the same time, in otherwords, a fluid flows from a pump through a manifold which distributesthe fluid to the culturing vessels evenly and simultaneously and loopsback to the pump. The fluid for use in the invention is referred to asculture medium. The term is also envisioned to mean that, in a mixedparallel and in series arrangement, at least one culturing vessel isconnected in parallel relative to the in series circuit containing twoor more culturing vessels connected in series. As shown in FIG. 2,culturing vessel 1 a contains adipose cells, vessel 1 b contains musclecells, vessel 1 c contains liver cells and vessel 1 d contains cardiaccells. The culturing vessels are connected to fresh medium vessel 2 andwaste vessel 3 by line 4. Medium is pumped through the line 4 into thevessels with pump 5. The medium flows from the pump to the manifold 7through line 4, then in parallel through vessel 1 a, 1 b, 1 c and 1 d.Valves 6 control the flow to the vessels, when the valve to the freshmedium vessel is open, the valve to the waste vessel is closed and visaversa.

It is envisioned in the present invention that each culturing vessel maycontain, localized differences in culture conditions. For example, thetemperature of the culturing vessels may be independently controlled tooptimize the growth conditions to the respective cell type culturedtherein. Another example is that the culture medium may be alteredlocally to include additives to optimize growth conditions or forselected expression. In this example, it is envisioned that the additivecan be added directly to the culturing vessel or can be added to thefluid feed line prior to entry into the culturing vessel.

As used herein, “vessels connected in series” means two or more vesselsare fed in series from a pump, in other words, a fluid flows from a pumpthrough a first culturing vessel, then into a second culturing vessel,etc. and ultimately is looped back to the pump. As was the case withparallel culturing vessels, each culturing vessel may contain localizeddifferences in culture conditions as described above. As shown in FIG.3, culturing vessel 1 a contains adipose cells, vessel, 1 b containsmuscle cells, vessel 1 c contains liver cells and vessel 1 d containscardiac cells. The culturing vessels are connected to fresh mediumvessel 2 and waste vessel 3 by line 4. Medium is pumped through thelines into the vessels with pump 5. The medium flows sequentially fromvessel 1 a to 1 b to 1 c to 1 d. Valves 6 control the now to thevessels, when the valve to the fresh medium vessel is open, the valve tothe waste vessel is closed and visa versa.

Within the parallel method, the in series method, or the mixed method,it is envisioned that a conditioned medium or a medium containingcultured cell mass may be obtained from each of the respective culturingvessels or even just from the final culturing vessel. It is alsoenvisioned that the conditioned medium may be recovered from the freshmedium vessel or from a recovery outlet or outlet port installed withinthe circuit. Shown in FIG. 4 is a combination of a parallel and an inseries system, culturing vessel 1 a contains adipose cells, vessel 1 bcontains muscle cells, vessel 1 c contains liver cells and vessel 1 dcontains cardiac cells. The culturing vessels are connected to freshmedium vessel 2 and waste vessel 3 by line 4. Medium is pumped throughthe lines 4 into the vessels with pump 5. The medium flows from the pumpto the manifold through line 4, then in parallel through vessel 1 a, 1 cand 1 d. Then the medium flows sequentially from vessel 1 a to 1 b.Valves 6 control the flour to the vessels, when the valve to the freshmedium vessel is open, the valve to the waste vessel is closed and visaversa.

As used herein, conditioned medium is medium harvested from culturedcells. It contains metabolites, growth factors, extracellular matrixproteins, cytokines, etc. secreted into the medium by the culturedcells. This selective collection allows tuning of the properties of theconditioned medium. For example, it may be envisioned that the firstculturing vessel is used to culture one cell type (e.g., muscle cells)and that the culture medium containing, for example cytokines and growthfactors, from that culturing vessel can be fed into the subsequentculturing vessel for growth of a second cell type (e.g., adipose cells)or a mixture of a second cell type and the first cell type (e.g.,adipose cells and muscle cells). This can be repeated in subsequentculturing vessels in series or in parallel. In each culturing, vessel itmay be desired to supplement fresh basal or growth, medium from, asecondary feed line. Of course, other nutrients, salts, hormones,factors, etc. may also be fed individually or in combinationindependently into each culturing vessel in the network. In thisembodiment it is envisioned that a membrane may be fitted betweensubsequent culturing vessels to ensure that no or very little cells passbetween culturing vessels.

Regardless of whether using the parallel method, the in series method orthe mixed method, conditioned medium can be obtained by having thecircuit closed (i.e., medium is allowed to flow through) to the freshmedium vessel and open (i.e., medium is prevented from flowing through)to the waste medium vessel and allowing the culture to grow withconstant circulation through the system for a time and under suitableconditions to reach the desired growth conditions (e.g. for meatapplications a desired cell density of at least 1×10⁷ cells/cc and forpersonal care products a certain composition related to metabolites,growth factors, and/or extracellular matrix proteins). In this method,the conditioned medium can be recovered from any location in the growthcircuit. Preferably, the conditioned medium is recovered from the freshmedium vessel or from a recovery outlet or outlet port included in thecircuit.

In a modification to the foregoing, it is envisioned that, at eitherpredetermined time intervals or based on reached established mediumbenchmarks (e.g., cell density or composition of the conditionedmedium), a percentage of the medium in the waste medium vessel isremoved and replaced with fresh basal medium added to the fresh mediumvessel and/or removed, purified, and returned to the fresh mediumvessel. For example, the percentage of medium to be exchanged can be atleast 2.5%, at least 5%, at least 7.5%, at least 10%, at least 12.5%, atleast 15%, at least 17.5%, at least 20% to at most 25%, at most 22.5%,at most 20%, at most 17.5%, at most 15%, at most 12.5% and all points,ranges and sub-ranges defined by these lower and upper limits. In thisvariant of the method, when purifying or replacing part of the wastemedium with fresh medium, the fresh medium vessel should be closed tomedia flow. This physically allows the addition of new medium into it,which is much more difficult if the liquid is flowing through it. Also,by closing the fresh medium vessel to liquid flow, all of the medium inthe system flows through the waste collecting vessel which facilitatesdraining and replacement in a controlled manner (e.g. let 2.5% out andput that 2.5% back into the fresh medium vessel).

In another modification to either of the foregoing, it is envisionedthat the waste medium vessel is connected to a dialyzer to filter outwaste from the medium and the treated medium is then returned to thesystem.

The culturing vessels may, independently, range in size from about 0.5liter to greater than 100,000 liters. In an embodiment of the presentinvention the culturing vessels independently range in size from 0.5liters to 250,000 liters, from 1 liters to 100,000 liters, from 2 litersto 50,000 liters, from 5 liters to 25,000 liters, from 10 liters to10,000 liters, from 25 liters to 5,000 liters, from 50 liters, to 2,500liters, from 100 liters to 1,000 liters; within this invention theranges are listed as merely exemplary and the lower limits and upperlimits may be selected to define alternative ranges.

Although not particularly limited, the number of culturing vessels mayrange from 1 to about 50. The range can have a lower limit of 2, 3, 4,5, 6, 7, 8, 9, and 10 and an upper limit of 45, 40, 35, 30, 25, and 20,inclusive of all ranges and sub-ranges and particular values that may bedefined by this particular values.

Suitable pumps are known in the art. Piping is used to connect the pumpto the vessels and the vessels to each other. Suitable piping is knownin the art and includes polyvinyl chloride (PVC), stainless steel,tubes, pipes, lines, hoes and the like. The piping may contain or beconnected to heating units.

The vessels may be provided with means for heating the fluid or inlineheating may be utilized. A separate vessel may be used for fresh medium;the fresh medium vessel may be connected to the system with a valve thatis open to provide fresh medium to the system when desired. Anotherseparate vessel may be used for waste collection; the waste vessel maybe connected to the system with a valve that is open to remove wastefrom the system when desired.

In general, the method described herein may be used to culture cells,and particularly muscle (e.g., smooth muscle) and fat cells to formengineered meat. The method includes providing a culture system,transferring basal medium or basal medium supplemented with non-animalderived growth factors and other components as might be needed for theefficient growth of cells, into the culturing, vessels, adding cells andculturing the cells to produce a conditioned medium. The basal medium(e.g. Dulbecco's Modified Eagle Medium; DMEM) may include water, salts,vitamins, minerals, amino acids and a carbon source such as glucose. Inan embodiment of the invention, the basal medium of the currentinvention does not include animal derived serum such as fetal bovineserum, calf serum or horse serum. As used herein, by “does not includeanimal serum” or “animal serum-free” is meant that the medium containsless than about 1% or less than about 0.5% or less than about 0.1% orless than about 0.01% or zero animal derived serum by total weight ofthe medium. It is envisioned within the invention that the serum-freemedium may contain growth factors and other substances, but nothingderived from an animal.

At least one type of cell is added to each culturing vessel. Suitabletypes of cells include but are not limited to muscle, fat, cartilage,liver, heart, kidney, lung, endothelial and combinations thereof. Othertypes of mammalian cells may also be used within the present invention.Suitable cells may be obtained by biopsy from fish, pig, cows, chicken,turkey, sheep, goat and the like. The various cell types, or theircombination, in the separate culturing vessels, connected through commoncirculation secrete cell and tissue specific growth factors andcytokines into the basal medium thus rendering it conditioned.

The various specialized primary animal cells, such as muscle or fat tobe added and subsequently expanded in the culturing vessels may beobtained via biopsy from live animals. Alternatively, the starter cellsmay be stem cells of various origin, such as satellite cells or inducedpluripotent stem cells (iPS cells) and culture conditions may beadjusted to develop these stem cells into the desired cell types. Thestarter cells in the individual culturing vessels may also begenetically modified cells such as immortalized cells allowing forunlimited number of cell divisions without any change in cell behavior.

The method of the present invention may include utilizing gases tooptimize growth conditions independently in each culturing vessel orthroughout the entirety of the system. Suitable gases include but arenot limited to oxygen, carbon dioxide and the like.

In one embodiment, the culture system is used to culture muscle, fat andcartilage cells. Salts are used to optimize growth conditions for cells.Suitable salts include but are not limited to those of sodium,potassium, calcium and the like. The amount of salt used is consistentwith ranges known in the art of tissue or cell culture. Cells neednutrients to grow; nutrients provide a source of carbon. Suitable carbonsources include but are not limited to glucose, glycerol, galactose,hexose, fructose, pyruvate, glutamine and the like. The amount of carbonsource used is consistent with ranges known in the art of tissue or cellculture. The basal medium may also include buffer such asphosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane (TRIS),phosphate-citrate buffer, sorensen's phosphate butler, sodium citratebuffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) andthe like. Alternatively, carbon dioxide can be fed into the medium tocontrol the pH. The pH is maintained at about 5.5 to about 7.5. Vitaminsare used to optimize growth conditions for cells. Suitable vitaminsinclude but are not limited to folic acid, nicotinamide, riboflavin, B₁₂and the like. The amount of vitamins used is consistent with rangesknown in the art of tissue or cell culture. Therefore, as stated above,the localized culture conditions can be independently controlled tooptimize the growth of the cells within the respective culturingvessels.

Culture conditions can be further controlled by temperature. Even thoughmammalian cells are typically cultured at body temperature, that is at37° C., sometimes deviation from this temperature might be desirable,depending on cell type. Thus the culturing vessels may be individuallytemperature controlled in the range of 20-38° C. (from room temperatureto near body temperature). However, exemplary conditions may includeincubating the culturing vessels to a temperature ranging from 20° C. to45° C., 32° C. to 42° C. from 35° C. to 40° C., from 36° C. to 38° C.,from 36.5° C. to 37.5° C., or 37° C., inclusive of all points, rangesand sub-ranges bound by the identified lower and upper limits. Furthercontrol and optimization of culturing can be achieved by the adjustmentof the perfusion, its speed, pressure and, in case of pulsatile flow,its pulse frequency and strength.

In another embodiment, the basal medium contains water, sodium chloride,HEPES buffer and cells. The cells are muscle, fat and cartilage cellsisolated from a biopsied tissue from livestock or poultry or stem cells(such as induced pluripotent stem cells, iPS cells or others). Thebiopsied tissue is digested to separate the individual cells. The cellsare then loaded into the culturing vessels of the above-describedculture system and cultured in the circulating basal medium or, ifneeded, initially, basal medium supplemented with plant based growthfactors. The cells are then cultured (e.g., incubated at 37° C. or atemperature independently controlled for the localized optimization ofgrowth in a culture and culture conditions preferred for the cell type).

As the cells are cultured they start producing cell and tissue specificgrowth factors and cytokines (as well as waste) and secrete them intothe common circulation, with this rendering the medium conditioned.Conditioned medium is basal medium supplemented with growth factors,hormones and other biochemical molecules secreted by cultured cells. Asused herein, basal medium is an unsupplemented growth medium used toculture microorganisms which do not need special nutrients. Basal mediumtypically includes water, salts, vitamins, minerals, amino acids and acarbon source such as glucose. Examples of growth factors include, butare not limited to fibroblast growth factors (FGF) and vascularendothelial growth factors (VEGF).

Consistent with the foregoing, the vessels in this system may be largecapacity three dimensional bioreactors from about 0.5 liter to about250,000 liters (or larger), the latter allowing to grow cell mass withtrillions of cells with biomass in thousands of kg. The use of thesebioreactors may enable the use of non adherent cells or adherent cellsgrown on floating microcarriers. An example of microcarriers that may beuseful in the present invention is taught in U.S. Pat. No. 9,752,122,the disclosure of which is hereby incorporated by reference.

In another embodiment it maybe more advantageous to use multiplecircuits with smaller bioreactors in each (for example 5 L), to mitigatethe damage from possible contamination. This way possible contaminationin one circuit does not lead to detrimental consequences.

In another embodiment the adherent cells are cultured in a stirredculturing vessel. Stirring makes the cells adhere to each other thusforming aggregates of increasing size. The substrate for the growth ofthe adherent cells thus is provided by the surface of the aggregates.With the increase of the size of the aggregate cells deep inside of theaggregates eventually become deprived of the culture medium: theaggregates develop a necrotic core. Since the biomass in the culturingvessels eventually is used for cultured meat products, necrotic cellsare still useful. The above outlined approach allows the culturing ofadherent cells in three dimensional bioreactors for the purposes ofcultured meat without the need for specific microcarriers.

The method of the present invention utilizes several connected culturingvessels to simultaneously grow various types of cells, each releasinggrowth factors and nutrients. In an exemplary embodiment, when connectedculturing vessels are utilized, the number of connected culturingvessels may range from about 2 to about 50. For example, the range canhave a lower limit of 2, 3, 4, 5, 6, 7, 8, 9, and 10 and an upper limitof 45, 40, 35, 30, 25, and 20. For example, in one embodiment, threeconnected vessels are culturing vessels, connected to a fresh mediumvessel, and a waste vessel and a pump in line to move medium between theculturing vessels. The culturing vessels may be perfused by basalmedium. The vessels are maintained at a sufficient temperature tosustain growth and continuously agitated. For example, one of theculturing vessels may contain liver cells, another muscle cells and thethird may contain adipose cells. In this exemplary embodiment of thepresent invention, the three culturing vessels may be arranged inparallel or in series relative to each other. It is also possible toestablish a mixed parallel in series arrangement wherein, for example,the culturing vessel containing muscle cells and the culturing vesselcontaining adipose cells are arranged in series, while the culturingvessel containing liver cells is arranged in parallel with respect tothe in series arrangement. A similar 4 culturing vessel example isexemplified in FIG. 3. It should be noted that the specific placement ofthe in series portion and the parallel portion is not limited. Forexample, the in series portion may be, relative to the pump, prior toall parallel circuits, between parallel circuits, or after the parallelcircuits. It is also envisioned that each circuit may be simultaneouslyfed by the pump.

The pump may transfer basal medium from the fresh medium vessel andcirculate the medium from one vessel to the next and so on. Suitablepumps include but are not limited to piston pumps and peristaltic pumps.Waste medium may be removed gradually from the system and replaced byfresh basal medium as needed. Fresh basal medium flows into the pumpcompartment and from there is circulated through the different culturingvessels. The cells are retained in their respective culturing vessels.To prevent cells from flowing to another culturing vessel, filters maybe used in line. Suitable filters are known in the art. Waste may beeliminated from the system.

A percentage of the conditioned medium may be removed from the circuitinto the waste vessel and fresh basal medium may be added. For example,10-15% of the circulating medium maybe be replaced daily. As freshmedium fills the system, the valve to the fresh medium reservoir is open(the valve to the waste reservoir is closed). As medium is circulatedthrough the system (mimicking blood flow), the cells in the varioustissues grow in numbers. At the same time the various specialized cells(heart muscle, hepatocytes, adipocytes, myoblasts) secrete growthfactors, nutrients and the like into the circulation.

Each cell type secretes cell/tissue specific molecules and conditionsthe medium. The method may hold as many different types of cells forsimultaneous growth as there are culturing vessels. For example, ifthere are ten culturing vessels, it may be desirable to simultaneouslygrow from one to ten types of cells. This cell culturing system allowsthe simultaneous culturing and growth of several cell types. These cellssecrete specialized chemicals (e.g. growth factors) that support thegrowth of all the cell types in the system (akin to how the circulatorysystem in the body maintains the proper functioning of all the organs,tissues and cells) in the body. In such a system only the basic“nutrients” (i.e. the basal medium) needs to be delivered from theoutside. Such basal medium contains components, such as salts, minerals,glucose, etc. The expensive components are then produced by the cells inthe system akin to what happens in the organism. Just as the circulatorysystem in the organism assures the maintenance and growth of a largenumber of cells (the adult human body contains about 37 trillion cells),the above described system allows, similarly, the culturing of aa largenumber of cells in a semi-autonomous manner.

The control/optimization of the circulation/pumping of the cellculturing system may be controlled or automated via computers, pressuregauges, etc.

Following cell culturing the cells can be separated from the conditionedmedium containing the secreted growth factors, etc. Separation may beachieved by many well-known techniques including, but not limited to,centrifugation and filtration.

The conditioned medium (filtrate) may be useful for personal carecompositions and nutritional supplements. For personal carecompositions, the conditioned medium may be applied to the skin. Theconditioned medium may be made into a cream, a lotion, an ointment, agel and the like. For the nutritional supplements, the conditionedmedium may be purified and ingested. The conditioned medium may be madeinto a liquid, a tablet, a gel, a powder and the like.

The personal care compositions may provide formulations suitable fortopical application to skin. The composition may further include acosmetically-acceptable carrier. The cosmetically-acceptable carrier maycomprise from about 50% to about 99%, by weight, of the composition(e.g., from about 80% to about 95%, by weight, of the composition). Thecompositions may be made into a wide variety of product types thatinclude but are not limited to liquid compositions such as lotions,creams, gels, sticks, sprays, shaving creams, ointments, cleansingliquid washes and solid bars, pastes, powders, mousses, masks, peels,make-ups, and wipes. These product types may comprise several types ofcosmetically acceptable carriers including, but not limited tosolutions, emulsions (e.g., microemulsions and nanoemulsions), gels,solids and liposomes. The following are non-limitative examples of suchcarriers. Other carriers can be formulated by those of ordinary skill inthe art.

The topical compositions useful in the present invention can beformulated as solutions. Solutions typically include an aqueous solvent(e.g., front about 50% to about 99% or from about 90% to about 95% of acosmetically acceptable aqueous solvent). Topical compositions may beformulated as a solution comprising an emollient. Such compositionspreferably contain from about 2% to about 50% of an emollient(s). Asused herein, “emollients” refer to materials used for the prevention orrelief of dryness, as well as for the protection of the skin. A widevariety of suitable emollients are known and may be useful in thepersonal care compositions. See International Cosmetic IngredientDictionary and Handbook, eds. Wenninger and McEwen, (The Cosmetic,Toiletry, and Fragrance Assoc., Washington, D.C., 7.sup.th Edition,1997) (hereinafter “CTFA Handbook”) which contains numerous examples ofsuitable materials.

A lotion can be made from such a solution. Lotions typically comprisefrom about 1% to about 20% (e.g., from about 5% to about 10%) of anemollient(s) and from about 50% to about 90% (e.g., from about 60% toabout 80%) of water.

Another type of product that may be formulated from a solution is acream. A cream typically comprises from about 5% to about 50% (e.g.,from about 10% to about 20%) of an emollient(s) and from about 45% toabout 85% (e.g., from about 50% to about 75%) of water.

Yet another type of product that may be formulated from a solution is anointment. An ointment may comprise a simple base of animal or vegetableoils or semi-solid hydrocarbons. An ointment may comprise from about 2%to about 10% of an emollient(s) plus from about 0.1% to about 2% of athickening agent(s). A more complete disclosure of thickening agents orviscosity increasing agents useful herein can be found in the CTFAHandbook.

The personal care compositions may be formulated as emulsions. If thecarrier is an emulsion, from about 1% to about 10% (e.g., from about 2%to about 5%) of the carrier comprises an emulsifier(s). Emulsifiers maybe nonionic, anionic or cationic. Suitable emulsifiers are disclosed in,for example, the CTFA Handbook.

Lotions and creams can be formulated as emulsions. Typically suchlotions comprise from 0.5% to about 5% of an emulsifier(s). Such creamswould typically comprise from about 1% to about 20% (e.g., from about 5%to about 10%) of an emollient(s); from about 20% to about 80% from 30%to about 70%) of water; and from about 1% to about 10% (e.g., from about2% to about 5%) of an emulsifier(s).

Single emulsion skin care compositions, such as lotions and creams, ofthe oil-in-water type and water-in-oil type are well-known in thecosmetic art and are useful for the personal care compositions.Multiphase emulsion compositions, such as the water-in-oil-in-water typeare also useful. In general, such single or multiphase emulsions containwater, emollients, and emulsifiers as essential ingredients.

The personal care compositions of this invention can also be formulatedas a gel (e.g., an aqueous gel using a suitable gelling agent(s)).Suitable gelling agents for aqueous gels include, but are not limitedto, natural gums, acrylic acid and acrylate polymers and copolymers, andcellulose derivatives (e.g., hydroxymethyl cellulose and hydroxypropylcellulose). Suitable gelling agents for oils (such as mineral oil)include, but are not limited to, hydrogenated butylene/ethylene/styrenecopolymer and hydrogenated ethylene/propylene/styrene copolymer. Suchgels typically comprise between about 0.1% and 5%, by weight, of suchgelling agents.

The personal care compositions useful in the subject invention maycontain, its addition to the aforementioned components, a wide varietyof additional oil-soluble materials and/or water-soluble materialsconventionally used in compositions for use on the skin at theirart-established levels.

The personal care compositions may be applied as needed and/or as partof a regular regimen ranging from application once a week up to one ormore times a day (e.g., twice a day). The amount used will vary with theage and physical condition of the end user, the duration of thetreatment, the specific compound, product, or composition employed, theparticular cosmetically-acceptable carrier utilized, and like factors.

Alternatively, a new batch of cells may be introduced into the circuit,such as muscle, fat and cartilage cells. The cells are then incubated ata suitable temperature and conditions for as long as needed. (Primarymammalian cells can divide a large number of times, the number ofdivisions set by the Hayflick limit. Alternatively, primary cells may beimmortalized that allows unlimited number of divisions.)

When the intent of the method is to produce a composition for use inbeing made into food products (e.g., a meat product) the cell growthdensity is monitored and the biomass is grown for a time and underconditions suitable to reach a cell density of at least 1×10⁷ cells/cc,of at least 5×10⁷ cells/cc, 1×10⁸ cells/cc, of at least 5×10⁸ cells/ccis reached. Exemplary conditions include incubating the culturingvessels to a temperature ranging from 20° C. to 45° C., 32° C. to 42°C., from 35° C. to 40° C., from 36° C. to 38° C. from 36.5° C. to 37.5°C., or 37° C., inclusive of all points, ranges and sub-ranges bound bythe identified lower and upper limits. Exemplary incubation times arefrom 10 to 40 hours, from 16 to 36 hours, from 20 to 30 hours, from 22to 28 hours, or from 24 to 26 hours inclusive of all points, ranges andsub-ranges bound by the identified lower and upper limits.

The biomass collected from the culture system may be made directly intofood such as a pate, hot dog and the like. Alternatively, it may bemixed with plant based ingredients such as soy bean (in particular,sprouted soy), potato and the like to produce a mixed product andlowering production costs. The amount of biomass in the mixed productmay range from about 50% to about 100% or from about 60% to about 90% orfrom about 70% to about 80%, etc. by weight, based on the total weightof the composition. More complex products, such as bacon, charcuterie,sausage, etc. may be engineered using methods of tissue engineering.Additional ingredients may be added to enhance texture, flavor andfragrance to the meat product. The biomass may be placed into a mold toproduce a desired shape, such as meat loaf.

The collected cultured medium from the waste vessel can be purified,filtered and reused. Alternatively, it can be centrifuged and theconcentrate may also be useful for personal care compositions andnutritional supplements. For personal care compositions, the concentratemay be applied to the skin. The concentrate may be made into a cream, alotion, an ointment, a gel and the like. For the nutritionalsupplements, the concentrate, appropriately processed, may be ingested.The concentrate may be made into a liquid, a tablet, a gel, a powder andthe like.

As a representation of the present invention is the followingembodiments:

-   [1] A cell culture system comprising:

a pump; at least two culturing vessels; a fresh basal medium vessel; anda waste collecting vessel, wherein the culturing vessels, the freshbasal medium vessel and the waste collecting vessel are connected inparallel with means for enabling a serum-free medium to move between thevessels.

-   [2] The system of [1], wherein each culturing vessel contains a    different type of cells selected from the group consisting of    muscle, fat, cartilage, liver, heart, kidney, and lung and other    mammalian cells.-   [3] The system of [1], wherein a single culturing vessel contains    multiple types of cells wherein the cells are selected from the    group consisting of muscle, fat, cartilage, liver, heart, kidney,    and lung, and other mammalian cells.-   [4] A method for making conditioned medium comprising:

providing the system of [1] with a serum-free medium;

adding animal cells into the culturing vessels;

circulating the serum free medium between the vessels;

heating the system to a temperature suitable for culturing; and

agitating and incubating the cells in the vessels for a time sufficientto culture the cells to a desired cell density.

-   [5] The method of [4], wherein each culturing vessel contains a    different type of cells selected from the group consisting of    muscle, fat, cartilage, liver, heart, kidney, and lung, and other    mammalian cells.-   [6] The method of [4], wherein a single culturing vessel contains    multiple types of cells wherein the cells are selected from the    group consisting of muscle, fat, cartilage, liver, heart, kidney,    and lung, and other mammalian cells.-   [7] A method for making meat comprising:

providing the system of [1] with a serum-free medium;

adding animal cells into the culturing vessels;

circulating the serum-free medium between the vessels;

heating the system to a temperature suitable for culturing;

agitating and incubating the cells in the vessels for a time sufficientto culture the cells to a desired cell density;

separating the cells from the medium; and

forming the cells into meat.

-   [8] The method of [7], wherein each culturing vessel contains a    different type of cells selected from the group consisting of    muscle, fat, cartilage, liver, heart, kidney, and lung, and other    mammalian cells.-   [8] The method of [7], wherein a single culturing vessel contains    multiple types of cells wherein the cells are selected from the    group consisting of muscle, fat, cartilage, liver, heart, kidney,    and lung, and other mammalian cells.-   [10] A cell culture system comprising:

a sub-system comprising a pump, a fresh basal medium vessel, and a wastecollecting vessel,

-   -   wherein the the fresh basal medium vessel and the waste        collecting vessel are connected in parallel with respect to each        other; and

at least three culturing vessels

-   -   wherein at least two of the at least three culturing vessels are        arranged in series relative to each other and at least one of        the at least three culturing vessels are arranged in parallel        relative to the culturing vessels arranged in series.

-   [11] A method for making meat comprising:

providing a cell culture system comprising a pump; at least oneculturing vessel; a fresh basal medium vessel; and a waste collectingvessel, wherein the pump, the culturing vessels, the fresh basal mediumvessel and the waste collecting vessel are connected with means forenabling a serum-free medium to move between the vessels;

adding animal cells into the culturing vessel;

circulating the serum-free medium between the vessels;

heating the system to a temperature suitable for culturing;

agitating and incubating the cells in the vessel for a time sufficientto culture the cells to a desired cell density;

separating the cells from the medium; and

forming the cells into meat.

-   [12] The method of [11], wherein the cells are selected from the    group consisting of muscle, fat, cartilage, liver, heart, kidney,    lung, and other mammalian cells and combinations thereof.-   [13] The method of [11], wherein the culturing vessels are connected    in series.-   [14] The method of [11], wherein the culturing vessels are connected    in parallel.-   [15] The method of [11], wherein the cell culture system comprises a    sub-system and at least three culturing vessels, wherein the cell    culture system is arranged such that

the sub-system comprises a pump, a fresh basal medium vessel, and awaste collecting vessel,

-   -   wherein the fresh basal medium vessel and the waste collecting        vessel which are connected in parallel with respect to each        other; and

with respect to the at least three culturing vessels at least two of theat least three culturing vessels are arranged in series relative to eachother and at least one of the at least three culturing vessels isarranged in parallel relative to the culturing vessels arranged inseries.

In the context of the present description, all publications, patentapplications, patents and other references mentioned herein, if nototherwise indicated, are explicitly incorporated by reference herein intheir entirety for all purposes as if fully set forth, and shall beconsidered part of the present disclosure in their entirety.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. In case of conflict, thepresent specification, including definitions, will control.

When an amount, concentration, or other value or parameter is given as arange, or a list of upper and lower values, this is to be understood asspecifically disclosing all ranges formed from any pair of any upper andlower range limits, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the present, disclosure be limited to thespecific values recited when defining a range.

Further, unless otherwise explicitly stated to the contrary, when one ormultiple ranges or lists of items are provided, this is to be understoodas explicitly disclosing any single stated, value or item in such rangeor list, and any combination thereof with any other individual value oritem in the same or any other list.

When the term “about” is used, it is used to mean a certain effect orresult can be obtained within a certain tolerance, and the skilledperson knows how to obtain the tolerance. When the term “about” is usedin describing a value or an end-point of a range, the disclosure shouldbe understood to include the specific value or end-point referred to.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of elements is notnecessarily limited to only those elements but can include otherelements not expressly listed or inherent to such process, method,article, or apparatus.

Further, unless expressly stated to the contrary, “or” and “and/or”refers to an inclusive and not to an exclusive. For example, a conditionA or B, or A and/or B, is satisfied by any one of the following: A istrue (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

The use of “a” or “an” to describe the various elements and componentsherein is merely for convenience and to give a general sense of thedisclosure. This description should be read to include one or at leastone and the singular also includes the plural unless it is obvious thatit is meant otherwise.

The above written description of the invention provides a manner andprocess of making and using it such that any person skilled in this artis enabled to make and use the same, this enablement being provided inparticular for the subject matter of the appended claims, which make upa part of the original description.

As used herein, the phrases “selected from the group consisting of,”“chosen from,” and the like include mixtures of the specified materials.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skilled in the art that references to a structure or featurethat is disposed “adjacent” another feature may have portions thatoverlap or underlie the adjacent feature.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements, these features/elements should not be limitedby these terms, unless the context indicates otherwise. These terms maybe used to distinguish one feature/element from another feature/element.Thus, a first feature/element discussed below could be termed a secondfeature/element, and similarly, a second feature/element discussed belowcould be termed a first feature/element without departing from theteachings of the present invention.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of, values),+/−10% of the stated value (or range of values), etc. Any numericalrange recited herein is intended to include all sub-ranges subsumedtherein.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

The above description is presented to enable a person skilled in the artto make and use the invention, and is provided in the context of aparticular application and its requirements. Various modifications tothe preferred embodiments will be readily apparent to those skilled inthe art, and the generic principles defined herein may be applied toother embodiments and applications without departing from the spirit andscope of the invention. Thus, this invention is not intended to belimited to the embodiments shown, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

EXAMPLES Example 1 Method of Making Conditioned Medium

In a culturing vessel, DMEM and cells obtained by needle biopsy of apig, cow, etc. are mixed to create a suspension. Carbon dioxide andoxygen are fed into the culturing vessel. The solution is heated up to37° C. and agitated continuously for from 10 to 40 hours. Opticaldensity measurements are made to determine when the conditioned mediumis ready for use. Alternatively, the analytes in the medium may bemeasured using chromatography.

Example 2 Method of Making a Concentrate and a Filtrate

The solution from the Example 1 is separated into a fraction containingthe cells and a fraction containing growth factors and nutrients.Fractionation is accomplished by filtering the solution into aconcentrate containing the cells while the filtrate will contain thegrowth factors and nutrients.

Example 3

The filtrate from Example 2 containing growth factors and nutrients isuseful for personal care applications such as anti-wrinkle serums, skinpigmentation serums, hydrating serums, and the like. Alternatively, theconcentrate may also be used in personal care applications.

Example 4 Method of Making Meat Product

The concentrate from the Example 2 is useful for food applications suchas pate, soups and the like. The concentrate can be dried further inmolds to make steaks, bacon, meatballs and the like. Suitable dryingmethods may include but are not limited to baking, pulling vacuum, airdrying, air frying, and the like. Additional ingredients may be added toenhance texture, flavor and fragrance to the food.

In this example, the biomass that is needed for the meat products comesfrom centrifuging the content of the culturing vessels. For example, thecontents of one culturing vessel containing muscle, fat and chondrocytecells, may be centrifuged to provide a biomass with the three differentcell types.

Example 5 Method of Making Nutritional Supplements

The concentrate from Example 2 is freeze dried and pulverized into apowder and filled into a pill capsule. The powder may also be mixed withwater, milk and/or fruits to make a beverage.

Numerous modifications and variations on the present invention arepossible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the accompanying claims, theinvention may be practiced otherwise than as specifically describedherein.

1. A cell culture system comprising; a pump; at least two culturingvessels; a fresh basal medium vessel; and a waste collecting vessel,wherein at least two culturing vessels, the fresh basal medium vesseland the waste collecting vessel are connected in parallel with means forenabling a serum-free medium to move between the vessels.
 2. The systemof claim 1, wherein each culturing vessel contains a different type ofcells selected from the group consisting of muscle, fat, cartilage,liver, heart, kidney, and lung, and other mammalian cells.
 3. The systemof claim 1, wherein a single culturing vessel contains multiple types ofcells wherein the cells are selected from the group consisting ofmuscle, fat, cartilage, liver, heart, kidney, and lung, and othermammalian cells.
 4. A method for making conditioned medium comprising:providing the system of claim 1 with a serum-free medium; adding animalcells into the culturing vessels; circulating the serum-free mediumbetween the vessels; heating the system to a temperature suitable forculturing; and agitating and incubating the cells in the vessels for atime sufficient to culture the cells to a desired cell density.
 5. Themethod of claim 4, wherein each culturing vessel contains a differenttype of cells selected from the group consisting of muscle, fat,cartilage, liver, heart, kidney, and lung, and other mammalian cells. 6.The system of claim 4, wherein a single culturing vessel containsmultiple types of cells wherein the cells are selected from the groupconsisting of muscle, fat, cartilage, liver, heart, kidney, and lung,and other mammalian cells.
 7. (canceled)
 8. The method of claim 13,wherein each of the at least two culturing vessels contains a differenttype of cells selected from the group consisting of muscle, fat,cartilage, liver, heart, kidney, lung, and other mammalian cells.
 9. Thesystem of claim 11, wherein the at least one culturing vessel containsmultiple types of cells, wherein the multiple types of cells areselected from the group consisting of muscle, fat, cartilage, liver,heart, kidney, lung, and other mammalian cells.
 10. (canceled)
 11. Amethod for making meat comprising: providing a cell culture systemcomprising a pump; at least one culturing vessel; a fresh basal mediumvessel; and a waste collecting vessel, wherein the pump, the culturingvessels, the fresh basal medium vessel and the waste collecting vesselare connected with means for enabling a serum-free medium to movebetween the vessels; adding animal cells into the culturing vessel;circulating the serum-free medium between the vessels; heating thesystem to a temperature suitable for culturing; agitating and incubatingthe cells in the vessel for a time sufficient to culture the cells toform aggregates of a desired cell density; separating the cells from themedium; and forming the cells into meat.
 12. The method of claim 11,wherein the animal cells are selected from the group consisting ofmuscle, fat, cartilage, liver, heart, kidney, lung, and other mammaliancells and combinations thereof.
 13. The method of claim 11, comprisingat least two culturing vessels, wherein the at least two culturingvessels are connected in series.
 14. The method of claim 11, comprisingat least two culturing vessels, wherein the at least two culturingvessels are connected in parallel.
 15. The method of claim 11, whereinthe cell culture system comprises a sub-system and at least threeculturing vessels, wherein the cell culture system is arranged such thatthe sub-system comprises a pump, a fresh basal medium vessel, and awaste collecting vessel, wherein the fresh basal medium vessel and thewaste collecting vessel which are connected in parallel with respect toeach other; and with respect to the at least three culturing vessels atleast two of the at least three culturing vessels are arranged in seriesrelative to each other and at least one of the at least three culturingvessels is arranged in parallel relative to the culturing vesselsarranged in series.
 16. The method of claim 11, comprising circulatingthe serum-free medium between the vessels, wherein the circulating isfor perfusion of the medium.
 17. The method of claim 11, wherein thedesired cell density is at least 1×10⁷ cells/cc (cubic cm) of themedium.
 18. The method of claim 11, wherein the volume of the at leastone culturing vessel is at least 0.5 liter.
 19. The method of claim 11,wherein the cells are grown without a microcarrier.
 20. A method formaking meat comprising: providing a cell culture system comprising apump; at least one culturing vessel; a fresh basal medium vessel; and awaste collecting vessel, wherein the pump, the culturing vessel, thefresh basal medium vessel and the waste collecting vessel are connectedwith means for enabling a culture medium to move between the vessels;adding animal cells into the culturing vessel; circulating the culturemedium between the vessels; heating the system to a temperature suitablefor culturing; agitating and incubating the cells in the vessel for atime sufficient to culture the cells to form aggregates of a desiredcell density; separating the cells from the medium; and forming thecells into meat.